Novel indolocarbazole derivatives potentially useful for the treatment of dementias characterized by tau hyperphosphorylation [Alzheimer""s disease (AD), frontal lobe degeneration (FLD), argyrophilic grains disease, subacute sclerotising panencephalitis (SSPE) as a late complication of viral infections in the CNS], and cancer.
Several dementias, most importantly Alzheimer""s disease (AD), are characterized by the formation of intracellular aggregates consisting of the microtubule-associated protein tau, termed neurofibrillary tangles (NFT). The importance of this biochemical abnormality for the clinical syndrome of dementia is illustrated by essentially three facts: (I) there is a close correlation between the state of dementia and the extent and density of NFT in various parts of the cortex [e.g., Bancher C. et al. (1993) Neurosci. Lett. 162, 179-182)]; (ii) individual neurons containing NFT in the cell body and/or the neurites are morphologically degenerating, i.e., lose synaptic connections and eventually die [Braak E. et al. (1994) Acta Neuropathol. 87, 554-567; Callahan L. M. et al., (1995) Neurobiol. Aging 16, 311-314]; (iii) a certain density of NFT in various otherwise unrelated dementias is always associated with dementia, without exception.
The tau protein contained in NFT is severely hyperphosphorylated [Goedert M. et al. (1995) Neurobiol. Aging 16, 325-334; Hasegawa M. et al. (1996) FEBS Lett. 384, 25-30]. This abnormal phosphorylation renders the protein incompetent to retain its original function, i.e., stabilization of the microtubule cytoskeleton, which is of fundamental importance for the integrity of a neuron [Iqbal K. et al. (1994) FEBS Lett. 349, 104-108; Garver T. D. et al. (1996) J. Neurosci. Res. 44, 12-20]. This explains-the paucity of intact microtubules in AD brains. Phosphorylation alone is responsible for this effect, as dephosphorylation restores the abilities of tau.
Because of a relationship between tau phosphorylation, cytoskeletal destabilization, synaptic loss and neuronal degeneration, and ultimately dementia, it would be therapeutically desirable to have pharmaceutical means to interfere with the pathological process of tau hyperphosphorylation.
The characteristics of hyperphosphorylated tau in NFT suggest that the protein kinase ERK2 is responsible for the pathological tau modification in AD [Drewes G. et al. (1990) EMBO J. 11, 2131-2138; Roder H. M. et al. (1993) Biochem. Biophys. Res. Commun. 193, 639-647]. ERK2 may exist in an abnormally activated state in AD [Roder H. M. et al. (1995) J. Neurochem. 64, 2203-2212). Inhibition of ERK2 has therefore been suggested as a point of interference to prevent tau hyperphosphorylation, and ultimately to stop NFT formation in neurons.
AD-like tau hyperphosphorylation can be induced in several cellular models (including brain slices), converting tau into a phosphorylation state indistinguishable from tau phosphorylated by ERK2 in vitro. The most convincing cellular models involve PP2A inhibition [Sautixc3xa9r, P. E. et al., Neurodegeneration 3, 53-60 (1994); Harris K. A. et al., Ann. Neurol. 33, 77-87 (1993)].
However, compounds which inhibit ERK2 and thereby prevent AD-like tau hyperphosphorylation in biological model systems, have previously not been disclosed. Such compounds can be expected to affect processes of neurofibrillary degeneration, tied to tau hyperphosphorylation, in a beneficial manner.
The protein kinases of the ERK family, often termed MAP-kinases, have also been implicated in a variety of important cellular regulation events outside the CNS, such as growth, differentiation and inflammation [e.g., Sale E. M. et al., EMBO J. 14, 674-684 (1995); Pages G. et al., Proc. Natl. Acad. Sci. USA 90, 8319-8323 (1993); Cowley S. et al., Cell 77, 841-852 (1994)]. Consequently, aberrant ERK activation has been implicated in several diseases characterized by loss of growth and differentiation control. In some tumors constitutive ERK activation is associated with cellular transformation due to dominant (activating) mutations in signal transduction proteins or viral proteins interfering with ERK inactivators [Sontag E. et al., Cell 75, 887-897 (1993); Leevers S. J. and Marshall C. J., EMBO J. 11, 569-574 (1992); Gallego G. et al., Proc. Natl. Acad. USA 89, 7355-7359 (1992); Gupta S. K. et al., J. Biol. Chem. 267, 7987-7990 (1992)].
The use of the disclosed kinase inhibitors for cancer is also indicated by their ability to inhibit cdc2 kinase. The role of cdc2 and homologous (cdks) kinases in cell cycle control is very well appreciated [Norbury C., and Nurse P., Annu. Rev. Biochem. 61, 441-470 (1992)]. Regulation of these enzymes is essential for both commitment to cell cycle from the resting state (START), and ordered transition through several phases of the cell cycle. The need for regulation is reflected in the existence of numerous positive and negative regulatory features of cdks, such as cyclin subunits, inhibiting (Thr) and activating (Tyr) phosphorylations, and endogenous peptide inhibitors.
Because of this central role of cdks in control of cell cycle and proliferation, they are considered as attractive drug targets for cancer therapies [e.g., Filguera de Azevedo W. et al., Proc. Natl. Acad. Sci. USA 93, 2735-2740 (1996)].
Indolocarbazole derivatives structurally related to the invention compounds have been described in the literature. The majority of these compounds are derived from the natural product K252a. The production and isolation of K252a was first published by Kase, et al. [J. of Antibiotics 39, 1059 (1986)]. Subsequent structure elucidation of K252a, b, c and d were reported in the same year by Yasuzawa et al. [J. of Antibiotics 39, 1072 (1986)]. Since the original disclosure and structure elucidation, K252a has been shown to be active in a variety of enzyme and cell-based assays. In particular, these compounds have demonstrated potent protein kinase C (PKC) activity. The most common uses claimed include: cancer, EP 0 323 171 (priority date Dec. 24, 1987), EP 0 643 966 (priority date Mar. 3, 1993), U.S. Pat. No. 4,923,986 (priority date Mar. 9, 1987), U.S. Pat. No. 4,877,776 (priority date Dec. 24, 1987), WO 94 27982 (priority date May 28, 1993); neurodegenerative disorders, WO 95 07911 (priority date Sep. 16, 1993), WO 94 02488 (priority date Dec. 24, 1992), antimicrobial [Prudhomme et al., J. Antibiotics 47, 792 (1994)], and hypertension [Hachisu et al., Life Sciences 44, 1351 (1989)]. 
In general, prior art compounds related to the invention are derived from K252a and contain the basic core structure where a tetrahydrofuran moiety is attached to the aglycone forming two glycosidic bonds. Modifications of the K252a core structure include additional substituents on the lactam and indole portions, and modifications of the a-hydroxy ester. The tetrahydrofuran oxygen in the core structure limits the opportunities for further modification.
Incorporation of a carbon at the tetrahydrofuran oxygen position of the K252a core structure significantly alters the core structure by removing the two glycosidic bonds and replacing the electron rich disubstituted atom with an electronically more neutral tetra-substituted moiety. This change also provides additional opportunities to incorporate functional groups that may enhance properties such as potency, selectivity, stability, toxicity, bioavailability, etc. which can result in an improved biological profile and consequently, a better therapeutic agent.
Compounds containing this important modification are completely inaccessible via synthetic methods used to prepare compounds of the prior art.
According to one aspect of the invention, a composition of matter is provided having the formula of Formula I, as follows: 
wherein Z is O or 2H (in which case the double bond is two single bonds),
R1 is H, OH, CO2R9, CONHR9, CH2OR9, or R9R10;
R2 is H or OH; R3 is H or OH; R4 is H or OH;
R5 is H, OH, NR9R10, NHCOR9, OCOR9, OCR9, halide, COOR9, or CONR9R10;
R6 is H, OH, NR9R10, NHCOR9, OCOR9, OCR9, halide, COOR9, or CONR9R10;
R7 is H, OH, O or halide;
R8 is H, OH, halide or nothing (when R7 is O);
R9 is an alkyl of 1-6 carbons, a cycloalkyl of 3-6 carbons or H;
R10 is an alkyl of 1-6 carbons, a cycloalkyl of 3-6 carbons or H.
In certain preferred embodiments, Z is O; R1 is OH, CO2R9, CHNHR9 or CH2OR9; R4 is H; R5 is H; R6 is H; and R8 is H. In other preferred embodiments, Z is O; R1 is CO2CH3 or CONHCH3; R2 is H; R3 is OH; R4 is H; R5 is H; and R6 is H. The most preferred compositions of matter are: 
According to another aspect of the invention, pharmaceutical compositions are provided. The pharmaceutical compositions include the compositions of matter described above, together with a pharmaceutically acceptable carrier. The preferred pharmaceutical compositions are as described above. Particularly preferred pharmaceutical compositions are those formulated in an oral dosage form.
In some embodiments, the pharmaceutical composition contains the composition of matter in an amount effective for inhibiting abnormal hyperphosphorylation associated with a dementia. In other embodiments, the pharmaceutical composition contains the composition of matter in an amount effective for inhibiting a cdk kinase, such as cdc2 kinase. In still other embodiments, the pharmaceutical composition contains the composition of matter in an amount effective to inhibit cell proliferation, and in certain embodiments to inhibit cancer cell proliferation by cancer cells expressing abnormal amounts of a cdk kinase.
According to another aspect of the invention, a method is provided for inhibiting in a subject a kinase which binds a compound of Formula I. A compound of Formula I is administered to a subject in need of such treatment in an amount effective to inhibit in the subject the kinase activity. Preferred compounds are as described above. In one embodiment, the subject has a dementia and the compound is administered in an amount effective to inhibit abnormal hyperphosphorylation characteristic of the dementia. The dementia can be, among other things, Alzheimer""s disease and the compound can be administered in an amount effective to inhibit phosphorylation activity of ERK2 which is characteristic of abnormal tau hyperphosphorylation in Alzheimer""s disease.
According to another aspect of the invention, a method is provided for treating a subject having a cancer which expresses abnormal levels of cdk kinase activity. The method involves administering to a subject in need of such treatment a compound of Formula I in an amount effective to inhibit the cdk kinase activity. In some embodiments, the kinase is cdc2 kinase. The preferred compounds are as described above.
According to another aspect of the invention, intermediates for preparing the compounds of Formula I are provided. The intermediates are described in detail in the text below. Particularly important intermediates are those numbered 13, 19 and 24.
According to still another aspect of the invention, a method is provided which involves the use of a compound of Formula I in the preparation of a medicament. In particular embodiments, the medicament is for treating a dementia (e.g. Alzheimer""s disease), a proliferative disorder (e.g. a cancer). These and other aspects of the invention are described in greater detail below.
According to another aspect of the invention, intermediates for manufacturing the above compounds are provided. These are compositions of matter comprising: 
wherein, Zxe2x95x90O or 2H (in which case the double bond is two single bonds); R11, R12=H, 
except that when R11 is not H, then R12 is H and when R12 is not H, then R11 is H;
R13, R13xe2x80x2=H or OPxe2x80x2, and R14 is H or OP. Preferably, Z is O and R13 and R13xe2x80x2 are H. Most preferable the composition of matter is compound 14. P is a protecting group. Preferred Ps for OP are benzyl- and t-butyl-dimethyl sylyl. Most preferably the composition of matter is compound 12, 13, 18, 19, 23 or 24.
Other compositions of matter are 
Wherein, Zxe2x95x90O or 2H; R13 and R13xe2x80x2=H or OP; and R14=O, H, OH or OP.
Preferably, Z is O and R13 and R13xe2x80x2 are H. Most preferably the composition of matter is compound 14.
Mixtures of the foregoing compounds including isomeric mixtures also are contemplated.